Transgenic animals for analyzing CYP3A4 cytochrome P450 gene regulation

ABSTRACT

The invention relates to the generation of non-human transgenic animals comprising a reporter construct for producing a detectable amount of a reporter molecule operably linked to a transcriptional regulatory nucleic acid molecule from the human CYP3A4 gene located between the initiation of transcription site of the gene and a position located 13,000 nucleotides upstream from the site. The invention also relates to the use of these animals for determining the effect of a compound, particularly, but not exclusively, a xenobiotic or steroid, on the regulation of expression of the CYP3A4 gene in a human.

TECHNICAL FIELD OF THE INVENTION

The invention relates to the generation of a transgenic animal and tothe use of the animal for determining the effect of a compound,particularly, but not exclusively, a xenobiotic or steroid, on theregulation of expression of a P450 gene in a human.

BACKGROUND OF THE INVENTION

Many endogenous and exogenous compounds are observed to have atherapeutic effect in drug development trials in vitro. However, theintended therapeutic effect is often not realised in clinical practice,for example, when compounds are co-administered, because certaincompounds induce the expression of the CYP3A4 gene. This inductiongenerates CYP3A4 cytochrome P450 molecules which metabolize compoundsbefore the intended therapeutic effect of each compound can be realised.Accordingly, induction of expression of the CYP3A4 gene interferes withintended dosage, leading to therapeutic failure or sub-optimaltreatment.

Induction of CYP3A4 gene expression is a significant problem for drugdevelopment because time, resources and expense are wanted in thedevelopment of candidate drugs for therapy of particular diseaseconditions which will ultimately fail or perform sub-optimally inclinical practice.

It would be advantageous to have an animal model for use in drugdevelopment trials from which, at an early stage of drug development,one could determine whether a candidate drug would be likely to achievean intended therapeutic effect in a human.

Such an animal model would not be useful unless at least some of theaspects of the regulation of CYP3A4 gene expression in the human,especially tissue specific expression, are reproduced. This is becausein the human, the CYP3A4 gene is expressed in specific tissues,including liver and small intestine, which many compounds inevitablycome into contact with when administered for the purpose of therapy.Accordingly, one would be unable to determine whether thebio-availability of a candidate drug would be sufficient for achievingan intended therapeutic effect in clinical practice in a model whichdoes not reproduce the constitutive and xenobiotic induced tissuespecific expression of the CYP3A4 gene that is observed in the human.

WO99/61622 and Goodwin et al. 1999 disclose a nucleic acid moleculelocated 8 kb upstream from the initiation of transcription site of theCYP3A4 gene which regulates transcription of the CYP3A4 gene in responseto xenobiotic compounds. These documents do not disclose elements forregulating the constitutive and xenobiotic inducible tissue specific anddevelopmental expression of the CYP3A4 gene observed in a human.

There is a need for an animal model which reproduces at least someaspects of the expression of the CYP3A4 gene in a human, for determiningwhether a compound, for example, one identified in a drug developmenttrial, would be likely to induce CYP3A4, and hence cause drug-druginteractions, or auto-induction of the metabolism of the drug understudy.

DESCRIPTION OF THE INVENTION

The invention seeks to address the above identified need and in a firstaspect provides a non-human mammal comprising:

(a) a regulatory nucleic acid molecule which is capable of regulatingtranscription of the human CYP3A4 gene and which comprises a nucleotidesequence that is identical to a sequence of the human CYP3A4 genelocated between the initiation of transcription site of the gene and aposition located at least 13,000 nucleotides upstream from the site; and

(b) a reporter nucleic acid molecule for producing a detectable amountof a reporter molecule for indicating regulation of transcription of thereporter nucleic acid molecule by the regulatory nucleic acid molecule

wherein the reporter and regulatory nucleic acid molecules are arrangedto permit the regulatory nucleic acid molecule to regulate transcriptionof the reporter nucleic acid molecule.

As described herein, the inventors have found that the incorporation ofa region of the human CYP3A4 gene that is located between the initiationof transcription site of the gene and a position 13,000 nucleotidesupstream of the initiation of transcription site into an animal modelprovides the animal with sufficient genetic information for reproducingthe constitutive and xenobiotic induced tissue specific expression ofthe CYP3A4 gene that is observed in humans. More specifically, theinventors have generated animal models which contain a transgenecomprising this region and have observed that these models provideconstitutive and xenobiotic inducible expression of a transgene in atissue pattern which reproduces the tissue specific expression of CYP3A4which is observed in a human. Importantly, the level of constitutiveexpression is sufficient to allow one to observe the effect on theregulation of tissue specific transgene expression, of administration ofa compound, for example, a xenobiotic or steroid, to the animal.

Further, the inventors have observed that the animal models describedherein also reproduce aspects of the constitutive and xenobioticinducible developmental expression of the CYP3A4 gene that is observedin humans.

These findings are unanticipated because prior to the invention, therewas no suggestion that the genetic information required for simulatingthe constitutive and xenobiotic induced tissue specific or developmentalexpression of the CYP3A4 gene that is observed in a human would becontained in the region of the human CYP3A4 gene between the initiationof transcription site of the gene and a position 13,000 nucleotidesupstream of the initiation of transcription site.

Further, prior to the invention, differences in the induction profile ofthe mouse CYP3A11 and the human CYP3A4 gene had been observed, anddifferences had also been observed in the ligand binding profile ofmouse transcription factors, especially PXR and CAR, and human PXR andCAR. Accordingly, there was no suggestion that a non-human animal wouldhave factors sufficient for interacting with a region of the CYP3A4 genefor reproducing the constitutive and xenobiotic induced tissue specificor developmental expression of CYP3A4 observed in a human.

Further, prior to the invention, mechanisms associated with transgeneintegration had been observed, such as gene silencing and mosaictransgene expression which limited the extent to which an atranscriptional enhancer element incorporated into a transgenic modelcould reproduce regulation of gene expression observed in a human.Accordingly, there was no suggestion that a region of the human CYP3A4gene would be capable of reproducing the regulation of expression of theCYP3A4 gene that is observed in a human. However, as described herein,the inventors have shown in 2 separate founder lines that the expressionof the transgene reproduces aspects of CYP3A4 gene expression that areobserved in humans.

Thus in a second aspect, the invention provides a non human mammalcomprising:

(a) a regulatory nucleic acid molecule comprising a nucleotide sequencethat is identical to the nucleotide sequence of the human CYP3A4 genethat extends about 13,000 nucleotides upstream from the initiation oftranscription site of the gene; and

(b) a reporter nucleic acid molecule for producing a detectable amountof a reporter molecule for indicating regulation of transcription of thereporter nucleic acid molecule by the regulatory nucleic acid molecule

wherein the reporter and regulatory nucleic acid molecules are arrangedto permit the regulatory nucleic acid molecule to regulate transcriptionof the reporter nucleic acid molecule.

In one embodiment, the regulatory nucleic acid molecule comprises thesequence shown in SEQ ID NO:1.

Further, as described herein, the inventors have generated transgenicanimals which contain a region of the human CYP3A4 gene between theinitiation of transcription site and a position about 3,200 nucleotidesupstream of the initiation transcription site and observed that thetransgene is not constitutively expressed or inducible by xenobiotics inthese animals. Accordingly, the inventors have found that the geneticinformation required for reproducing the constitutive and xenobioticinduced tissue specific and developmental expression of CYP3A4 observedin a human is contained in the region of the human CYP3A4 gene betweenthe position located about 3,200 nucleotides upstream of the initiationof transcription site of the gene and a position 13,000 nucleotidesupstream of the initiation of transcription site.

Thus, in a third aspect, the invention provides a non-human mammalcomprising:

(a) a regulatory nucleic acid molecule comprising a nucleotide sequencethat is identical to the sequence of the human CYP3A4 gene that extendsabout 8,000 nucleotides upstream from a position about 3,000 nucleotidesupstream from the initiation of transcription site of the gene; and

(b) a reporter nucleic acid molecule for producing a detectable amountof a reporter molecule for indicating regulation of transcription of thereporter nucleic acid molecule by the regulatory nucleic acid molecule

wherein the reporter and regulatory nucleic acid molecules are arrangedto permit the regulatory nucleic acid molecule to regulate transcriptionof the reporter nucleic acid molecule.

In one embodiment, the regulatory nucleic acid molecule comprises thesequence shown in SEQ ID NO:2.

In a fourth aspect, the invention provides a non-human mammalcomprising:

(a) a regulatory nucleic acid molecule which is capable of regulatingtranscription of the human CYP3A4 gene and which comprises a nucleotidesequence that is identical to the sequence of the human CYP3A4 gene thatextends about 600 nucleotides upstream from a position about 7,200nucleotides upstream of the initiation of transcription site of thegene; and

(b) a reporter nucleic acid molecule for producing a detectable amountof a reporter molecule for indicating regulation of transcription of thereporter nucleic acid molecule by the regulatory nucleic acid molecule

wherein the reporter and regulatory nucleic acid molecules are arrangedto permit the regulatory nucleic acid molecule to regulate transcriptionof the reporter nucleic acid molecule.

In one embodiment, the regulatory nucleic acid molecule comprises thesequence shown in SEQ ID NO:3.

In another embodiment, the regulatory nucleic acid molecule has thesequence of any one of the following fragments of the CYP3A4 gene:

(i) a fragment consisting of from nucleotide positions −13,000 to +53;

(ii) a fragment consisting of from nucleotide positions −13,000 to−12,700 contiguous with −8000 to +53;

(iii) a fragment consisting of from nucleotide positions −13,000 to−5,100 contiguous with −1,200 to +53;

(v) a fragment consisting of from nucleotide positions −7,800 to −6,000contiguous with −362 to +53;

(vi) a fragment consisting of from nucleotide positions −7,500 to −6,000contiguous with −362 to +53;

A regulatory nucleic acid molecule which has the sequence of a fragmentconsisting of from nucleotide positions −7836 to −7207 contiguous with−362 to +53 is particularly preferred, as this construct contains theminimal sequences necessary for regulating transcription of the humanCYP3A4 gene, more specifically, an element responsive to xenobiotics(the “Xenobiotic Response element Module” or “XREM”) and the proximalpromoter of the CYP3A4 gene.

The regulatory nucleic acid molecule of the invention typically containsat least one enhancer capable of regulating transcription of a humanCYP3A4 gene when contacted with a nuclear receptor. Examples of suchenhancers are those capable of regulating transcription of a humanCYP3A4 gene when contacted with a nuclear receptor bound to a ligand,such as a xenobiotic or steroid. Other examples are those capable ofregulating transcription of a human CYP3A4 gene when contacted with anuclear receptor consisting of a heterodimer of PXR (pregnane Xreceptor, otherwise known as SXR (steroid and xenobiotic receptor)) andRXR (9-cis retinoic acid receptor), or CAR (constitutive androstanereceptor-β) and RXR.

The inventors believe that certain nucleic acid molecules which havesubstantially the same nucleotide sequence as a regulatory nucleic acidmolecule of the invention would also have sufficient genetic informationfor reproducing the constitutive and xenobiotic induced tissue specificand developmental expression of the CYP3A4 gene that is observed in ahuman. Accordingly, it will be understood that nucleotides could bemodified or deleted in regions of the regulatory nucleic acid molecule,more specifically, those regions which do not contain an enhancer suchas those described above, without significantly limiting the capacity ofthe molecule to regulate transcription of the human CYP3A4 gene.

The inventors recognise that it would be advantageous to provide ananimal model further capable of reproducing the expression of otherhuman genes, specifically those genes encoding products which modify ormodulate the therapeutic activity of exogenous and endogenous compoundsused for therapy and cause drug-drug interactions, for example,cytochrome P450 genes or ABC transporter superfamily genes, for example,P-glycoprotein (otherwise known as MDR-1). The regions controlling theconstitutive and xenobiotic induced tissue specific expression of someof these genes are known, and in some instances, non-human animal modelshave been generated. The inventors recognise that the genetic backgroundof these animals could be incorporated into the non-human mammal of thepresent invention, for example, by conventional breeding techniques.

Thus in a fifth aspect, the invention provides a non-human mammal of anyone of the first to fourth aspects of the invention, further comprising:

(c) a further regulatory nucleic acid molecule which is capable ofregulating transcription of a human gene; and

(d) a further reporter nucleic acid molecule for producing a detectableamount of a further reporter molecule for indicating regulation oftranscription of the further reporter nucleic acid molecule by thefurther regulatory nucleic acid molecule

wherein the further reporter and further regulatory nucleic acidmolecules are arranged to permit the further regulatory nucleic acidmolecule to regulate transcription of the further reporter nucleic acidmolecule.

In one embodiment, the at least one further regulatory nucleic acidmolecule has a sequence shown in SEQ ID NO:4. In another embodiment, theat least one further regulatory nucleic acid molecule has a sequenceshown in SEQ ID NO:5.

Although the regulatory nucleic acid molecule of the invention describedherein is sufficient for reproducing the constitutive tissue specificand developmental expression of the CYP3A4 gene that is observed in ahuman, the inventors recognise that aspects of the xenobioticinducibility of the gene could be better reproduced in an animal byincorporating at least one human transcription factor that is capable ofinteracting with the regulatory nucleic acid molecule for regulatingtranscription of the human CYP3A4 gene. Examples of such factors arenuclear receptors. These receptors may be those capable of regulatingCYP3A4 gene transcription in a human when the receptor is bound to aligand, such as a xenobiotic or steroid. One example of such a receptoris the human PXR (pregnane X receptor, otherwise known as SXR (steroidand xenobiotic receptor)). Another suitable receptor is the human CAR(constitutive androstane receptor-β). Non-human animals comprising ahuman PXR or CAR receptor are known. The inventors recognise that thegenetic background of these animals could be incorporated into thenon-human mammal of the present invention, for example, by conventionalbreeding techniques.

Thus in a sixth aspect, the non-human animal of the invention furthercomprises at least one human transcription factor for regulatingtranscription of a human CYP3A4 gene. Preferably the transcriptionfactor is a nuclear receptor. Preferably, the nuclear receptor is aheterodimer of the human PXR (pregnane X receptor, otherwise known asSXR (steroid and xenobiotic receptor)) and human RXR (9-cis retinoicacid receptor) or human CAR (constitutive androstane receptor-β) andhuman RXR.

It follows that the reporter nucleic acid molecule can be any moleculewhich is capable of detection when the reporter nucleic acid molecule istranscribed. For example, the reporter nucleic acid molecule could bethe CYP3A4 cytochrome, or the mRNA transcript which is translated toproduce the cytochrome. Those reporter molecules which are commerciallyavailable, including firefly luciferase, β-galactosidase, alkalinephosphatase, green fluorescent protein or chloramphenicol acetyltransferase can be used.

Thus in one embodiment, the reporter nucleic acid molecule is capable ofproducing a reporter molecule selected from the group of reportermolecules consisting of firefly luciferase, β-galactosidase, alkalinephosphatase, green fluorescent protein or chloramphenicol acetyltransferase.

While the non-human mammal of the invention, as exemplified below, is amouse, the inventors believe that any other non-human mammal could beused in the invention, especially those for which standard transgenictechniques have been developed including for example, rat and rabbit.However, typically the non-human mammal is a mouse.

In another aspect, the invention provides a tissue of a non-human mammalof the invention.

In one embodiment, the tissue is an embryo capable of producing anon-human mammal of the invention.

In a further aspect, the invention provides a method of determiningwhether a compound is capable of effecting the transcription of a humanCYP3A4 gene the method comprising the following steps:

(a) administering the compound to a non human mammal according to theinvention and

(b) determining whether the reporter molecule is produced by thereporter nucleic acid molecule in the mammal.

In one embodiment, the production of the reporter molecule indicatesthat the binding compound is capable of effecting the transcription ofthe human CYP3A4 gene.

Any compound can be tested in the method however, preferred compoundsare xenobiotic or steroid compounds.

The inventors recognise that a non human animal which comprises a 5′flanking region of CYP3A4 gene but which is deficient for the regionfrom −7836 to −7207 would be useful as a negative control in a methodfor determining whether a compound is capable of regulatingtranscription of the human CYP3A4 gene.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. CYP3A4/lacZ transgene constructs used to generate transgenicmice. The upstream regions of the human CYP3A4 gene are depicted as openboxes with the position of the XREM at approximately −7.5 kb of theCYP3A4 gene indicated by cross-hatching. The 5′-flanking region extendedfrom 56 bp downstream of the transcription initiation site to a HindIIIsite at −3,213 in the construct designated −3CYP3A4/lacZ and to a KpnIsite at −12,926 kb in construct −13CYP3A4/lacZ. The coding region of theE. coli lacZ gene together with eukaryotic translational initiation andtermination signals, transcription termination and poly adenylationsites are indicated by a solid box.

FIG. 2. Xenobiotic induction of hepatic transgene expression. Femalemice from line 9/4 harbouring the −13CYP3A4/lacZ transgene were treatedwith various reagents. Histochemical staining of liver slices with X-galrevealed an increased zone of blue staining cells containingβ-galactosidase after treatment with rifampicin, phenobarbital andpregnenolone 16α-carbonitrile compared with corn oil treated mice.

FIG. 3. Comparison of the xenobiotic induction profile of the−13CYP3A4/lacZ transgene with the mouse Cyp3a11 gene. Transgenic micefrom line 9/4 were treated with a range of xenobiotic reagents andnaturally occurring steroids. A. Transgene expression was assessed bydetermining β-galactosidase activity in total liver lysates using theONPG assay. The units of β-galactosidase activity are given as A₄₂₀/mgliver/minute. Dexamethasone and pregnenolone 16α-carbonitrile were themost potent xenobiotic activators of the −13CYP3A4/lacZ transgene, whilerifampicin treatment resulted in relatively low levels. The steroidspregnenolone and 17α-progesterone were very weak inducers. B. Hepaticexpression of the endogenous mouse Cyp3a11 gene was examined in the samemice by Northern analysis. A similar pattern of induction to theCYP3A4/lacZ transgene was observed with both xenobiotic and endogenousregulators. The data are presented as the mean+/−the standard deviationfor 3 animals.

FIG. 4. Dose response of −13CYP3A4/lacZ transgene expression aftertreatment with dexamethasone. A. Male mice from line 9/4 were treatedwith from 1 to 100 mg/kg dexamethasone. Higher doses of dexamethasoneresulted in increased β-galactosidase activity (determined in liverlysates as described in FIG. 3). B. Zonal expansion of transgeneexpression with increasing doses of dexamethasone. X-gal staining offrozen liver sections revealed greater numbers of hepatocytes containingtransgene-derived β-galactosidase activity after treatment with 1, 10and 100 mg/kg dexamethasone. At low doses there are limited numbers oftransgene-expressing cells immediately adjacent to the central vein.With higher doses there are more cells committed to transgene expressionextending across the liver lobule towards the portal tract.

FIG. 5A-5E (SEQ ID NO: 1) Sequence of the CYP3A4 5′-flanking regionincluded in the −13 CYP3A4/lacZ construct. This sequence corresponds to−12,926 to +56 base pairs relative to the transcription initiation siteof the CYP3A4 gene.

FIG. 6A-6D (SEQ ID NO: 2) Sequence of the 5′-flanking region of theCYP3A4 gene extending from −12,926 to −3,213 base pairs and representingthe difference in sequence between the −13 CYP3A4/lacZ and the −3CYP3A4/lacZ constructs.

FIG. 7. (SEQ ID NO:3)The “Xenobiotic-Responsive Enhancer Module” (XREM)of the human CYP3A4 gene. This region encompasses −7836 to −7207 basepairs relative to the transcription initiation site of the CYP3A4 gene.

FIG. 8A-8C (SEQ ID NO: 4) The 5′-flanking region of the human CYP3A7gene (Genbank Accession No. AF329900). The extent of the sequences is−11,133 to +52 base relative to the transcription initiation site of theCYP3A7 gene.

FIG. 9A-9C (SEQ ID NO: 5) Sequence of the 5′-flanking region of thehuman MDR1 gene (p-glycoprotein gene) encompassing −10,000 to +200 basepairs relative to the transcription initiation site of the MDR1 gene.Sequence derived from within Genbank sequence Accession Number AC002457.

An embodiment of the invention is now described in the following Examplewhich will be understood to merely exemplify and not to limit the scopeof the invention.

EXAMPLE Materials and Methods

Transgene constructs. Two transgene constructs were synthesized with theupstream 5′ flank of the human cytochrome P450 CYP3A4 gene linked to theE. coli lacZ reporter gene (FIG. 1). The first construct, designated−3CYP3A4/lacZ, contained the region of the CYP3A4 gene from the HindIIIsite at −3213 bp relative to the transcription start site to nucleotide+56 bp downstream of the transcription start site. The other construct,designated −13CYP3A4/lacZ, included the region of the CYP3A4 gene fromthe KpnI site at −12,926 bp upstream to +56 bp downstream of thetranscription start site. It includes the DNA sequences of the XREMregion located between −7836 and −7208 in addition to the proximalpromoter of the CYP3A4 gene. The DNA sequence of the CYP3A4 gene between−10468 bp and +906 bp has been determined and deposited with theGenBank/EMBL/DDJB database under accession number AF185589. Additionalsequence information covering the region −10,469 bp to −12,926 bp wasobtained from publically accessible Genbank files. The E. coli lacZreporter gene comprises the coding region for the bacterial enzymeβ-galactosidase flanked by DNA sequences for eukaryotic translationalstart and stop signals, SV40 transcriptional termination andpolyadenylation signals and an intron. The CYP3A4/lacZ transgeneconstructs were released from vector sequences and purified on agarosegels prior to microinjection

Generation of transgenic mouse lines. Mice carrying the CYP3A4/lacZtransgenes were created by microinjection of the DNA constructs into thepro-nuclei of zygotes harvested from FVB/N strain mice. Microinjectionand manipulation of embryos were carried by standard techniques. Stabletransgenic mouse lines were established by breeding from transgenicfounders identified by Southern analysis. Administration of xenobioticsto mice. 8-10 week old male and female mice hemizygous for the−3CYP3A4/lacZ and −13CYP3A4/lacZ transgenes were used to test theability of a range of xenobiotics and hormones to activate expression oftransgene-derived β-galactosidase. Mice were administered the followingreagents and vehicles by single daily intraperitoneal injection for 4days: rifampicin/corn oil; dexamethasone phosphate/H₂O; pregnenolone16α-carbonitrile/2% Tween 20 in H₂O; phenobarbital/H₂O; clotrimazole/2%Tween 20; phenytoin/2% Tween 20; 17α-OH progesterone/2% Tween 20;pregnenolone/2% Tween 20. All reagents were supplied by Sigma ChemicalCo. (St Louis, Mo.) except for dexamethasone phosphate which wasobtained from Faulding (Mulgrave, Australia) and pregnenolone16α-carbonitrile from Upjohn Co. (Kalamazoo, Mich.). The dose used forall reagents to test for induction of the transgene was 100 mg/kg bodyweight. Dose response studies were carried out in the range of 1-100mg/kg with male hemizygous transgenic mice.

Analysis of transgene and mouse Cyp3a gene expression. β-galactosidaseactivity was visualised in slices and frozen sections of liver and othertissues by staining with X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Tissues were fixedin 0.25% glutaraldehyde, 0.1M phosphate buffer pH7.3, 5 mM EGTA, 2 mMMgCl₂: washed in 0.1M phosphate buffer pH7.3, 0.01% sodium deoxycholate,0.025% NP40, 2 mM MgCl₂ and stained by incubation at 37° C. in washsolution supplemented with 1 mg/ml X-gal, 5 mM potassium ferricyanide,and 5 mM potassium ferrocyanide. The level of β-galactosidase activitywas determined in whole liver homogenates [100 mg fresh tissue/ml 0.25MTris-HCl (pH 7.3)] using the O-nitrophenyl-β-D-galactopyranoside (ONPG)assay according to standard techniques. After appropriate dilution thehomogenate was incubated with β-galactosidase assay reagent (0.1M sodiumphosphate buffer (pH7.3)/1 mM MgCl₂/50 mmol β-mercaptoethanol/0.88 mg/mlONPG) at 37° C., quenched by the addition of 1M Na₂CO₃ and theabsorbance at 420 nm determined. The units of β-galactosidase activityare given as A₄₂₀/mg liver/minute.

The levels of endogenous mouse Cyp3a mRNA expression were determined byNorthern analysis using a riboprobe complementary to nucleotides852-1061 of the mouse Cyp3a11 cDNA. Filters were stripped and reprobedwith an 18S rRNA oligonucleotide to normalise loading.

Results

4 transgenic lines were generated with the construct containing the −3.2kb region of the human CYP3A4 gene linked to lacZ. Transgene-derivedβ-galactosidase activity was not detected in kidney, large and smallintestine, spleen, lung and liver tissue from mice for all 4−3CYP3A4/lacZ transgenic lines treated with vehicle or xenobiotics(Table 1). In contrast, transgene expression was readily detected in 3of the 4 lines carrying the −13CYP3A4/lacZ construct. Line 9/4 had avery low constitutive level in the liver, with β-galactosidase detectedonly in isolated hepatocytes adjacent to major blood vessels.Administration of xenobiotics resulted in robust expression in a zone ofcells surrounding the central vein (FIG. 2). As the basal level oftransgene expression in untreated mice in line 9/4 is extremely low,induction is obvious and is essentially an off/on process. Expression inother tissues in mice from line 9/4 was restricted to the gut,predominantly in the villi of the small intestine.

The relative degree of induction for a range of xenobiotics was analysedby determining the transgenic β-galactosidase activity in liver lysatesof mice from line 9/4 (FIG. 3A). Dexamethasone and pregnenolone16α-carbonitrile were the most potent inducers, while rifampicinactivated the transgene to relatively modest levels. Phenobarbital,clotrimazole and phenytoin were intermediate inducers. The inductionprofile of the transgene in line 9/4 was similar to that observed forthe endogenous Cyp3a11 gene in the same mice (FIG. 3B), likelyreflecting the activation profile of the mouse rather than the humanPXR. Activation of the transgene was observed with naturally occurringsteroids such as pregnenolone and 17α-progesterone, however theinduction was weak compared with xenobiotics. There was a marked genderdifference in hepatic transgene expression, with lower levels observedin females than in males for most reagents. Such a male-predominantpattern was not evident in the induction profile of the mouse Cyp3a11gene. Indeed higher levels of Cyp3a11 mRNA were observed in females thanmales after treatment with rifampicin and pregnenolone 16α-carbonitrile.The reason for this apparent reversal in gender-related transgeneexpression pattern is not known. However, as Cyp3a11 mRNA is only justdetectable in males of the FVB/N strain of mice, it may be attributed tothe relatively greater degree of induction of the mouse Cyp3a11 gene inmales compared to females (FIG. 3B).

The other line which showed significant transgene expression −15/10, hada higher constitutive level in both the liver and small intestine inuntreated mice. Expression was not detected in other organs, confirmingthe tissue specificity observed in line 9/4. The same set of reagentswere capable of increasing hepatic and intestinal transgene expressionto the same levels as in mice from line 9/4. However, the overall degreeof induction was not as great as observed in line 9/4 due to the higherbasal level in line 15/10. The induction profile was similar withdexamethasone being the most potent activator and rifampicin the least(data not shown).

Dose response of xenobiotic induction. The activation of transgeneexpression in line 9/4 by dexamethasone was dose-dependent over therange 1 to 100 mg/kg (FIG. 4A). The higher transgene-derivedβ-galactosidase activity in liver homogenates from mice treated withincreasing doses of dexamethasone was associated with an expanded zoneof cells which were stained by X-gal. At low doses of dexamethasone aring of hepatocytes only 1-2 cells thick around the central veinexpressed the transgene (FIG. 4B). With 100 mg/kg dexamethasone the zoneof X-gal positive hepatocytes increased to up to 10 cells, approximatelymidway between the central vein and the portal triad. A similardose-dependent expansion of hepatocytes expressing the transgene wasobserved with other reagents and also in line 15/10 which also containedthe −13CYP3A4/lacZ construct.

TABLE 1 Expression of CYP3A4/IacZ transgenic lines LIVER Small ConstructLine No. Copy No. Basal Inducible Intestine -3CYP3A4/ IacZ

13 24 31 39  15 >100  80  10 − − − − − − − − − − − − -13CYP3A4/ IacZ

13/5  9/4 9/7 15/10  70   5  50   8 − + − ++ − ++++ + ++++   + − ++++

1. A non-human mammal comprising: a regulatory nucleic acid moleculewhich is capable of regulating transcription of the human CYP3A4 geneand which comprises a nucleotide sequence that is identical to asequence of the human CYP3A4 gene located between the initiation oftranscription site of the gene and a position located at least 13,000nucleotides upstream from the site; and a reporter nucleic acid moleculefor producing a detectable amount of a reporter molecule for indicatingregulation of transcription of the reporter nucleic acid molecule by theregulatory nucleic acid molecule; wherein the reporter and regulatorynucleic acid molecules are arranged to permit the regulatory nucleicacid molecule to regulate transcription of the reporter nucleic acidmolecule.
 2. A non human mammal comprising: a regulatory nucleic acidmolecule comprising a nucleotide sequence that is identical to thenucleotide sequence of the human CYP3A4 gene that extends about 13,000nucleotides upstream from the initiation of transcription site of thegene; and a reporter nucleic acid molecule for producing a detectableamount of a reporter molecule for indicating regulation of transcriptionof the reporter nucleic acid molecule by the regulatory nucleic acidmolecule; wherein the reporter and regulatory nucleic acid molecules arearranged to permit the regulatory nucleic acid molecule to regulatetranscription of the reporter nucleic acid molecule.
 3. A mammalaccording to claim 2 wherein the regulatory nucleic acid moleculecomprises the sequence shown in SEQ ID NO:1.
 4. A non-human mammalcomprising: a regulatory nucleic acid molecule comprising a nucleotidesequence that is identical to the sequence of the human CYP3A4 gene thatextends about 8,000 nucleotides upstream from a position about 3,000nucleotides upstream from the initiation of transcription site of thegene; and a reporter nucleic acid molecule for producing a detectableamount of a reporter molecule for indicating regulation of transcriptionof the reporter nucleic acid molecule by the regulatory nucleic acidmolecule; wherein the reporter and regulatory nucleic acid molecules arearranged to permit the regulatory nucleic acid molecule to regulatetranscription of the reporter nucleic acid molecule.
 5. A mammalaccording to claim 4 wherein the regulatory nucleic acid moleculecomprises the sequence shown SEQ ID NO:2.
 6. A non-human mammalcomprising: a regulatory nucleic acid molecule which is capable ofregulating transcription of the human CYP3A4 gene and which comprises anucleotide sequence that is identical to the sequence of the humanCYP3A4 gene that extends about 600 nucleotides upstream from a positionabout 7,200 nucleotides upstream of the initiation of transcription siteof the gene; and a reporter nucleic acid molecule for producing adetectable amount of a reporter molecule for indicating regulation oftranscription of the reporter nucleic acid molecule by the regulatorynucleic acid molecule; wherein the reporter and regulatory nucleic acidmolecules are arranged to permit the regulatory nucleic acid molecule toregulate transcription of the reporter nucleic acid molecule.
 7. Amammal according to claim 6 wherein the regulatory nucleic acid moleculecomprises the sequence shown in SEQ ID NO:3.
 8. A non-human mammalcomprising: a regulatory nucleic acid molecule which is capable ofregulating transcription of the human CYP3A4 gene and which comprises anucleotide sequence of a fragment of the human CYP3A4 gene consisting offrom nucleotide positions −7836 to −7207 contiguous with −362 to +53;and a reporter nucleic acid molecule for producing a detectable amountof a reporter molecule for indicating regulation of transcription of thereporter nucleic acid molecule by the regulatory nucleic acid molecule;wherein the reporter and regulatory nucleic acid molecules are arrangedto permit the regulatory nucleic acid molecule to regulate transcriptionof the reporter nucleic acid molecule.
 9. A mammal according to claim 3,further comprising at least one human transcription factor forregulating transcription of a human CYP3A4 gene.
 10. A mammal accordingto claim 9 wherein the transcription factor is a nuclear receptor.
 11. Amammal according to claim 10 wherein the nuclear receptor is aheterodimer of the human pregnane X receptor and human 9-cis retinoicacid receptor or a heterodimer of human constitutive androstanereceptor-β and human 9-cis retinoic acid receptor.
 12. A mammalaccording to claim 3 wherein the reporter nucleic acid molecule iscapable of producing a reporter molecule selected from the group ofreporter molecules consisting of firefly luciferase, β-galactosidase,alkaline phosphatase, green fluorescent protein or chloramphenicolacetyl transferase.
 13. A mammal according to claim 3 wherein the mammalis a mouse.
 14. A tissue of a mammal according to claim
 3. 15. A tissueaccording to claim 14 wherein the tissue is an embryo.
 16. A method ofdetermining whether a compound is capable of effecting the transcriptionof a human CYP3A4 gene the method comprising the following steps:administering the compound to a non human mammal according to any one ofthe preceding claims; and determining whether the reporter molecule isproduced by the reporter nucleic acid molecule in the mammal.
 17. Amethod according to claim 16 wherein the production of the reportermolecule indicates that the binding compound is capable of effecting thetranscription of the human CYP3A4 gene.